Bidirectional Photochemistry of Antarctic Microbial Rhodopsin: Emerging Trend of Ballistic Photoisomerization from the 13-cis Resting State

The decades-long ultrafast examination of nearly a dozen microbial retinal proteins, ion pumps, and sensory photoreceptors has not identified structure–function indicators which predict photoisomerization dynamics, whether it will be sub-picosecond and ballistic or drawn out with complex curve-crossing kinetics. Herein, we report the emergence of such an indicator. Using pH control over retinal isomer ratios, photoinduced transient absorption is recorded in an inward proton pumping Antarctic microbial rhodopsin (AntR) for 13-cis and all-trans retinal resting states. The all-trans fluorescent state decays with 1 ps exponential kinetics. In contrast, in 13-cis it decays within ∼300 fs accompanied by continuous spectral evolution, indicating ballistic internal conversion. The coherent wave packet nature of 13-cis isomerization in AntR matches published results for bacteriorhodopsin (BR) and Anabaena sensory rhodopsin (ASR), which also accommodate both all-trans and 13-cis retinal resting states, marking the emergence of a first structure–photodynamics indicator which holds for all three tested pigments.

2 mL of the cell culture was used to inoculate 1 L of 2×YT media in presence of 100 µg/ml ampicillin (at 37°C, 130 rpm), and grown to an optical density OD 600 of 0.6. Overexpression was induced by 1 mM isopropyl-β-D-thiogalactoside (IPTG), followed by addition of 10 µM all-trans retinal, and additional shaking for 8 hrs. The cells were then collected by centrifugation at 7000 rpm and 4 °C for 15 min. The collected cell pellet was re-suspended in 50 mM MeS [2-(Nmorpholino)-ethanesulfonic acid] buffer pH 6, 300 mM NaCl, 5 mM imidazole and 5 mM MgCl 2 .The suspension was lysed with 0.2 mg/mL lysozyme overnight at 4°С in the presence of 2 μg/mL DNase and 1.5% (w/v) DDM (n-dodecyl-β-D-maltoside). DDM solubilized protein was separated as supernatant by centrifugation at 18000 rpm and 4°С for 35 min, and then loaded on Co 2+ -NTA resin (ThermoFisher scientific) pre-equilibrated with 50 mM MES buffer containing 300 mM NaCl, 5 mM Imidazole at pH 6. Other protein contaminants were removed by washing with 50 mM MES buffer containing 300 mM NaCl, 50 mM Imidazole, and 0.06% DDM, at pH 6.
Protein elution was done with an elution buffer (50 mM Tris-HCl, 300 mM NaCl, 200 mM Imidazole, 0.06% DDM; pH 7.5). The desired purified protein was obtained followed by washing off the imidazole by using an Amicon Ultra centrifugal filter tube (10kDa MWCO) by three times repeated solvent exchange with 0.02% DDM and 100 mM NaCl. The concentrated AntR was stored in 0.06% DDM with 100 mM NaCl for further use. For the spectroscopic measurements, AntR was dissolved in Tris buffer at pH 8 with 0.06% DDM, and 100 mM NaCl or Sodium acetate buffer of pH 3.4 (with 0.06% DDM, 100 mM NaCl).
TA measurements: TA measurements were performed using a homemade flow cell with a 0.25 mm path length equipped with 0.15 mm glass windows. A syringe pump is employed for flowing, and its speed is tuned so that consecutive laser pulses excite fresh AntR, and signals are not dependent on increase of flow rate. Sample integrity is monitored consistently by taking absorption spectra throughout the measurement. A hybrid multipass amplified Ti-sapphire laser system that generates 30 fs, 1 mJ pulses at ~800 nm with a 1000 Hz repetition rate was used for TA measurement. Details of the detection set-up and supercontinuum probe generation are detailed elsewhere. S2 The pump pulses are generated on a home-built noncollinear optical parametric amplifier (NOPA), generating tunable broad-band pulses between 500-700 nm wavelength. The NOPA output is compressed using a pair of chirp mirrors (LASER QUANTUM, UK) and a 19channel deformable mirror coupled to a grating compressor. S3 The temporal profile of NOPA output is characterized by Frequency gated optical grating method (SI figure S1). Probing in the NIR is performed using the same laser set-up but at a reduced 370 Hz repetition rate. The supercontinuum probe pulses are generated using a fraction of 800 nm fundamental output on a 3 mm sapphire crystal. The dispersed probe pulses are detected on an InGaAs NIR sensor (B&W TEK, USA). Otherwise, set-up is as used for the visible measurements. Figure S1: (a) 2D colour map of FROG for the pump 1. X-axis represents wavelength, the y-axis represents the group delay, and transmitted intensity is colour coded; (b) The pump 1 spectrum and group delay obtained from FROG. The left y-axis (black) stands for normalized pump intensity, and the right y-axis (red) represents group delay.

Impulsive vibration:
Excitation with ultrashort pulse generates coherent wavepacket motion in the ground and excited state, which is detected in the TA data as a periodic modulation at the early delay.

Absorption spectra of retinal isomers:
A dilute sample of AntR (OD 555  0.085) was made to react with 400 mM hydroxylamine solution at pH 8 for 15 min, under light irradiation with a Schott 250 W cold light source (with a long-pass >520 nm) cut-off filter. The reaction with hydroxylamine produced retinal oxime characterized by a blue-shifted absorption band at ~365 nm (figure S3a). From a difference absorption spectrum obtained between before and after the hydroxylamine reaction, as shown in figure S3b, the ratio of ΔOD 555 (for retinal pigment) and ΔOD 365 (for retinal oxime) was estimated.
AntR sample at pH 3.4 contains both isomers, but at pH 8 almost exclusively, the all-trans isomer is present. Therefore, the 13-cis AntR absorption spectrum could be isolated if its absorption maximum and difference with the all-trans state are known. We evaluated the absorption maximum of 13-cis state from C=C stretch and absorption maximum frequency maximum at 525 nm. On the other hand, light-dependent bi-stability at pH 6 is assigned to the isomeric composition variation. Under blue light (< 480 nm) illumination, the absorption maximum is located at 547 nm, but as the light turned to red (> 560 nm), the maximum shifted to 542 nm. Such a redshift in the absorption maximum is assigned to the increased all-trans fraction. S1 Hence, the absorption difference between blue and red (blue -red) light irradiation represents alltrans minus 13-cis state spectrum ( Figure S4a). Therefore, using prior knowledge of absorption maximum absorption difference with all-trans, we evaluated 13-cis state absorbance (figure 5a main text).

S4 b
). An acidic sample contains about 60 ± 5% 13-cis and 40 ± 5 % all-trans retinal. Figure S4 b shows the identical absorbance of individual isomers at the pump wavelength maximum.
Therefore, pump 1 excites individual isomers to the same extent. As a result, the finite difference (discussed in the main text and figure 3) estimated a 35 ± 5 % abundance of all-trans retinal in the pH 3.4. The absorption spectrum of the 13-cis state (figure s4 b) will be used for determining its photoisomerization quantum yield later. Figure S4: (a) Difference spectra between the all-trans and 13-cis states; (b) Absorption spectra of pH 3.4 AntR along with its all-trans and 13-cis components. We used an ~ 100 fs actinic pulse with a wavelength centred at 560 nm to excite ~12 ± 2 % of the all-trans ground state (Table S2) faster than all-trans. Therefore, the all-trans component in the VVV data is reduced to 78 ± 3 % of VV using dynamic difference spectra. Figure S6: (a) 2D colour map of TA data (VVV) of dark-adapted AntR at pH 8 in the presence of actinic excitation as described in Eq S2. The X and Y axes represent probe wavelength ( p ) / wavenumber ( p ) and delay between pump and probe (t), respectively. The time axis is linear for the first 0.5 ps, followed by a logarithmic scale from 0.5 -1.5 ps. OD colour-coding is depicted in the attached scale; (b) 2D colour map of TA data (VV) of dark-adapted AntR at pH 8 in the absence of actinic excitation as described in Eq S1. All axes of (b) are the same as (a). On the other hand, VV data shows pronounced vibrionic modulation near 530 nm probe wavelength due to Raman vibrations of the all-trans manifold. However, the amplitude of such modulation is reduced for VVV data set. Interestingly, a comparison of vibronic modulation also yields an identical reduction of all-trans components from VV to VVV. Therefore, the Raman vibration of the all-trans manifold could be used as a quantitative marker. The retinal's transition dipole is positioned along the linear retinal backbone, causing TA anisotropy of 0.4, identical to the theoretical estimate. Therefore, we need to consider the effect of the photo selection because of the specially oriented sample. S8 The primary pump excites a homogeneous population. We are probing with an identical polarization of light after the excitation with a linearly polarised light need cos 2  special integration. Similarly, a three-pulse measurement consisting of excitation, re-excitation and probing with the same electric field polarization requires cos 4  special integration.

Photochemistry of the K intermediate and quantum efficiency of all-trans 15-anti
We assume the following: 1) Only the actinic pump will include depletion effects 2) Even in the actinic interaction, the depletion will only be introduced by an average intensity assumed uniform in sample depth. Absorption crosssection at 560 nm () m -2 % excited at the magic angle % excited at the identical pump and probe polarization (6.4 ±0.5) × 10 18 (1.9 ±0.1) × 10 -20 12 ± 2 22 ± 4 % excitation at magic angle = 100*e (-J) ; % excitation at identical pump and probe polarization = % excitation at magic angle×(9/5); Our pump-probe stage is the basis for interrogation with a comparison with and without the actinic interaction. We know that the actinic pump-probe leads to a 22 ± 4% S 0 bleach just after the pump.
So: Or A = 0.43 ± 0.7 The cos 2 factor relates to the probing interaction, with the integral in the denominator representing the probe signal without actinic depletion. By running these integrals with various values of A we can find the correct factor representing J and assuming unity quantum efficiency.
A value of A=0.43 provided the right depletion measure.
on the signal in a secondary pump-probe sequence: . This results in the Dynamic difference or vibronic modulation measure shows 22 ± 3 % reduction of all-trans state concentration following actinic excitation. Therefore, isomerization efficiency is 85 ± 15 %.

Isomerization quantum efficiency of 13-cis 15-syn to all-trans 15-syn:
Experimentally, isomerization quantum yield determination of the 13-cis resting-state remained challenging due to the absence of a pure 13-cis sample. In general, the ground state all-trans isomer of MRP has red-shifted absorption and higher absorbance than its 13-cis isomer. Therefore, alltrans state has a higher transition dipole than is 13-cis isomer. Since transition dipole is proportional to the integrated extinction coefficient (), the  ratio between all-trans and 13-cis states will provide the dipole strength ratio. Knowing the individual absorption spectra, calculated transition dipole ratio between all-trans and 13-cis states is 1.18 : 1 of ground state AntR ( figure   S9). Performing similar analysis on the absorption spectra of other MRP's like BR and ASR provides identical dipole strength ratio. S9,S10 Figure S9: (a) Abortion spectrum of all-trans AntR (black line) and the highlighted area is integrated. Integration from the start of the absorption (15400 cm -1 ) to 22000 cm -1 provides a value of 1.9 x 10 8 L.mol -1 .cm -2 . (b) The absorption spectrum of 13-cis AntR and its integration.
In order to make a viable comparison same 6600 cm -1 width from the start of the absorption is integrated. Integration of 13-cis state absorption is 1.6 x 10 8 L.mol -1 .cm -2 .
Photoexcited 13-cis 15-syn resting-state isomerized to all-trans 15-syn (K') ground state within sub 100 fs. The TA spectra of 13-cis isomer at a longer t show a more positive OD portion than its negative part, indicating transition dipole increases following isomerization. Hence, the K' spectrum is red-shifted with an increased absorbance like an all-trans resting state. We assume that the transition dipole ratio between all-trans and 13-cis is invariant, and its value remains 1.18 : 1. According to our assumption, the transition dipole ratio between K' and 13-cis must be 1.18 : 1.
To calculate the quantum efficiency of isomerization, number of excited chromophores and the number of those isomerized are required. Knowing the density of excitation and absorption, crosssection (table S3) excitation fraction is calculated and absorption is presented in figure s10 a. Next, from the integrated area of the TA spectra at 200 ps, assuming transition dipole ratio of all-trans and 13-cis isomer, reacted 13-cis absorbance is presented in figure s10 b. Comparing these two absorption spectra yields a quantum efficiency of 50 ± 15 %. Absorption cross-section at 545 nm () m -2 % excited at the magic angle % excited at the identical pump and probe polarization (4.5 ±0.5) × 10 18 (1.3 ±0.1) × 10 -20 5 ± 1 9 ± 2 Figure S10: (a) 13-cis state absorbance calculated from absorption cross-section and photon density; (b) TA spectra of 13-cis state at 200 ps (black) and reacted 13-cis state (red) absorption.
Since the TA spectra at t = 200 ps is integrated from 14000 to 23000 cm -1 , ratio of areas between all-trans and 13-cis states spectra to be used for 13-cis absorbance separation. Integrated area ratio between all-trans and 13-cis states for such range is 1.23. Next, using the integrated area of TA difference spectra at 200 ps and integrated area ratio between all-trans and 13-cis states of 1.23, reacted 13-cis absorbance is isolated.